1. Field of the Invention
The invention relates to long-wavelength fluorescent proteins (FPs) and their uses.
2. Background Information
The initial discovery and characterization of the green fluorescent protein (GFP) from Aequorea victoria opened up numerous opportunities for downstream development and use. That discovery resulted in intensive research to identify homologous proteins with different optical and chemical properties from naturally occurring sources. Since these proteins are naturally fluorescent and are able to autocatalytically create the fluorophore from their amino acids in the chain, they can be fused to other proteins to provide substantially any polypeptide as a labeled fusion product. This has provided a variety of opportunities for determining events in cells, preparing reagents for assays and other purposes, and detecting interactions between proteins.
There are many properties of the new FPs (“FPs”) that affect their usage. Among these many aspects are responses to changes in the environment, such as pH and salt concentration and the effect of illumination (photobleaching and photosensitization), and the like. Inherent characteristics of the FPs include excitation and emission wavelength, the number of peaks, quantum efficiency, extinction coefficient, Stokes shift, time to maturation, and ability to participate in fluorescence resonance energy transfer, among other properties. It is also found that the FPs differ in their degree of aggregation, where some remain monomeric, and others are involved in dimeric or higher orders of aggregation. In addition, proteins can be mutated to varying degrees to provide new capabilities, where the change in composition results in different physical or optical properties, as compared to the natural FP.
Some characteristics, depending on the context may be advantageous, while others may be disadvantageous. For example, color-shifts during maturation of an expressed protein can be used to track gene expression through time. Other properties, such as photobleaching can be applied to detect cellular events.
It appears that the variety of FPs now known to exist in corals, sea anemones, and hydromedusae can be traced back to a single ancestral gene. A number of domains are shared by different FPs. The fluorophore appears to be derived from a triplet of x-Y-G (65-67), where x varies significantly. Most of the FPs have a “_-can” structure comprised of β-sheets. The amino acid side chains protruding into the _-can in which the fluorophore is enclosed affect the optical properties of the FP.
In view of the extensive variation in properties between different FPs, there is substantial interest in identifying new naturally occurring FPs and in modifying the sequences to introduce new capabilities or diminish undesirable properties. The high diversity of organisms with FPs makes it an arduous task to identify, purify and isolate specific FPs having interesting properties.